Two alternative conformations of mRNA in the human ribosome during elongation and termination of translation as revealed by EPR spectroscopy.

The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin-spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation.

Tumor Cell-Specific 2'-Fluoro RNA Aptamer Conjugated with Closo-Dodecaborate as A Potential Agent for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a binary radiotherapeutic approach to the treatment of malignant tumors, especially glioblastoma, the most frequent and incurable brain tumor. For successful BNCT, a boron-containing therapeutic agent should provide selective and effective accumulation of 10B isotope inside target cells, which are then destroyed after neutron irradiation. Nucleic acid aptamers look like very prospective candidates for carrying 10B to the tumor cells. This study represents the first example of using 2'-F-RNA aptamer GL44 specific to the human glioblastoma U-87 MG cells as a boron delivery agent for BNCT. The closo-dodecaborate residue was attached to the 5'-end of the aptamer, which was also labeled by the fluorophore at the 3'-end. The resulting bifunctional conjugate showed effective and specific internalization into U-87 MG cells and low toxicity. After incubation with the conjugate, the cells were irradiated by epithermal neutrons on the Budker Institute of Nuclear Physics neutron source. Evaluation of the cell proliferation by real-time cell monitoring and the clonogenic test revealed that boron-loaded aptamer decreased specifically the viability of U-87 MG cells to the extent comparable to that of 10B-boronophenylalanine taken as a control. Therefore, we have demonstrated a proof of principle of employing aptamers for targeted delivery of boron-10 isotope in BNCT. Considering their specificity, ease of synthesis, and large toolkit of chemical approaches for high boron-loading, aptamers provide a promising basis for engineering novel BNCT agents.

A Versatile Solid-Phase Approach to the Synthesis of Oligonucleotide Conjugates with Biodegradable Hydrazone Linker.

One of the ways to efficiently deliver various drugs, including therapeutic nucleic acids, into the cells is conjugating them with different transport ligands via labile or stable bonds. A convenient solid-phase approach for the synthesis of 5'-conjugates of oligonucleotides with biodegradable pH-sensitive hydrazone covalent bonds is proposed in this article. The approach relies on introducing a hydrazide of the ligand under aqueous/organic media to a fully protected support-bound oligonucleotide containing aldehyde function at the 5'-end. We demonstrated the proof-of-principle of this approach by synthesizing 5'-lipophilic (e.g., cholesterol and α-tocopherol) conjugates of modified siRNA and non-coding RNAs imported into mitochondria (antireplicative RNAs and guide RNAs for Mito-CRISPR/system). The developed method has the potential to be extended for the synthesis of pH-sensitive conjugates of oligonucleotides of different types (ribo-, deoxyribo-, 2'-O-methylribo-, and others) with ligands of different nature.

Knockdown of the mRNA encoding the ribosomal protein eL38 in mammalian cells causes a substantial reorganization of genomic transcription.

The ribosomal protein eL38 is a component of the mammalian translation machine. The deletion of the Rpl38 locus in mice results in the Tail-short (Ts) mutant phenotype characterized by a shortened tail and other defects in the axial skeleton development. Here, using the next-generation sequencing of total RNA from HEK293 cells knocked down of eL38 mRNA by transfection with specific siRNAs, we examined the effect of reduced eL38 content on genomic transcription. An approximately 4-fold decrease in the level of eL38 was shown to cause changes in the expression of nearly 1500 genes. Among the down-regulated genes, there were those responsible for p53 activity, Ca2+ metabolism and several signaling processes, as well as genes involved in the organization and functioning of the cytoskeleton. The genes related to rRNA processing and translation, along with many others, including those whose dysregulation is associated with developmental disorders, turned out to be up-regulated. Thus, we demonstrated that the decreased RPL38 expression leads to a significant reorganization of genomic transcription. Our findings suggest a possible link between the balance of eL38 and genes implicated in osteogenesis, thereby contributing to the elucidation of the reasons for the appearance of the above Ts mutant phenotype in animals.

Terminal Mono- and Bis-Conjugates of Oligonucleotides with Closo-Dodecaborate: Synthesis and Physico-Chemical Properties.

Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, siRNA components, and antisense agents. Particularly, the closo-dodecaborate anion represents a high-boron-containing residue with remarkable chemical stability and low toxicity, and is suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of closo-dodecaborate attached to the 5'-, 3'-, or both terminal positions of DNA, RNA, 2'-O-Me RNA, and 2'-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of closo-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of closo-dodecaborate residues at the 3'-end of all oligomers stabilized their complementary complexes, whereas analogous 5'-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2'-O-methyl and 2'-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized the DNA/DNA duplex but gave a strong stabilization of the duplex with an RNA target. According to circular dichroism spectroscopy results, two terminal closo-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from the B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.

Aptamers for Proteins Associated with Rheumatic Diseases: Progress, Challenges, and Prospects of Diagnostic and Therapeutic Applications.

Nucleic acid aptamers capable of affine and specific binding to their molecular targets have now established themselves as a very promising alternative to monoclonal antibodies for diagnostic and therapeutic applications. Although the main focus in aptamers' research and development for biomedicine is made on cardiovascular, infectious, and malignant diseases, the use of aptamers as therapeutic or diagnostic tools in the context of rheumatic diseases is no less important. In this review, we consider the main features of aptamers that make them valuable molecular tools for rheumatologists, and summarize the studies on the selection and application of aptamers for protein biomarkers associated with rheumatic diseases. We discuss the progress in the development of aptamer-based diagnostic assays and targeted therapeutics for rheumatic disorders, future prospects in the field, and issues that have yet to be addressed.

G-quadruplex 2'-F-modified RNA aptamers targeting hemoglobin: Structure studies and colorimetric assays.

Biosensors that rely on aptamers as analyte-recognizing elements (also known as aptasensors) are gaining in popularity during recent years for analytical and biomedical applications. Among them, colorimetric ELISA-like systems seem very promising for biomarker detection in medical diagnostics. For their development, one should thoroughly consider the characteristics of the aptamers, with a particular focus on the secondary structure. In this study, we performed an in-depth structural study of previously selected hemoglobin-binding 2'-F-RNA aptamers using CD spectroscopy, enzymatic probing, and specific fluorophore binding. Only a combination of different assays allowed us to prove G-quadruplex formation for anti-hemoglobin 2'-F-RNA aptamers. We also demonstrated a possible application of these 2'-F-RNA aptamers for microplate colorimetric detection of human hemoglobin in both direct and sandwich formats.

Postsynthetic On-Column 2' Functionalization of RNA by Convenient Versatile Method.

We report a universal straightforward strategy for the chemical synthesis of modified oligoribonucleotides containing functional groups of different structures at the 2' position of ribose. The on-column synthetic concept is based on the incorporation of two types of commercial nucleotide phosphoramidites containing orthogonal 2'-O-protecting groups, namely 2'-O-thiomorpholine-carbothioate (TC, as "permanent") and 2'-O-tert-butyl(dimethyl)silyl (tBDMS, as "temporary"), to RNA during solid-phase synthesis. Subsequently, the support-bound RNA undergoes selective deprotection and follows postsynthetic 2' functionalization of the naked hydroxyl group. This convenient method to tailor RNA, utilizing the advantages of solid phase approaches, gives an opportunity to introduce site-specifically a wide range of linkers and functional groups. By this strategy, a series of RNAs containing diverse 2' functionalities were synthesized and studied with respect to their physicochemical properties.

Knockdown of the Ribosomal Protein eL29 in Mammalian Cells Leads to Significant Changes in Gene Expression at the Transcription Level.

An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.

Novel Convenient Approach to the Solid-Phase Synthesis of Oligonucleotide Conjugates.

A novel and convenient approach for the solid-phase 5'-functionalization of oligonucleotides is proposed in this article. The approach is based on the activation of free 5'-hydroxyl of polymer support-bound protected oligonucleotides by N,N'-disuccinimidyl carbonate followed by interaction with amino-containing ligands. Novel amino-containing derivatives of closo-dodecaborate, estrone, cholesterol, and α-tocopherol were specially prepared. A wide range of oligonucleotide conjugates bearing closo-dodecaborate, short peptide, pyrene, lipophilic residues (cholesterol, α-tocopherol, folate, estrone), aliphatic diamines, and propargylamine were synthesized and characterized to demonstrate the versatility of the approach. The developed method is suitable for the conjugate synthesis of oligonucleotides of different types (ribo-, deoxyribo-, 2'-O-methylribo-, and others).

Exploring the interactions of short RNAs with the human 40S ribosomal subunit near the mRNA entry site by EPR spectroscopy.

The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3'-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2'-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated.

Bioluminescent aptamer-based sandwich-type assay of anti-myelin basic protein autoantibodies associated with multiple sclerosis.

Bioluminescent solid-phase sandwich-type microassay was developed to detect multiple sclerosis (MS)-associated autoantibodies in human sera. The assay is based on two different 2'-F-Py RNA aptamers against the target autoantibodies as biospecific elements, and Ca2+-regulated photoprotein obelin as a reporter. The paper describes elaboration of the assay and its application to 91 serum samples from patients with clinically definite MS and 86 ones from individuals healthy in terms of MS. Based on the receiver-operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers sensitivity of 63.7% and specificity of 94.2%. The area under the ROC curve (AUC) value of 0.87 shows a good difference between the groups under investigation. The likelihood ratio of 10.97 proves the diagnostic value of the assay and its potential as one of the laboratory MS-tests.

Complexes of Pro-Apoptotic siRNAs and Carbosilane Dendrimers: Formation and Effect on Cancer Cells.

This paper examines the complexation of anti-cancer small interfering RNAs (siRNAs) by cationic carbosilane dendrimers, and the interaction of the formed complexes with HeLa and HL-60 cancer cells. Stepwise formation of the complexes accompanied by the evolution of their properties has been observed through the increase of the charge ratio (dendrimer/siRNA). The complexes decrease the viability of both "easy-to-transfect" cells (HeLa) and "hard-to transfect" ones (HL-60), indicating a high potential of the cationic carbosilane dendrimers for siRNA delivery into tumor cells.

Hydrogels of Polycationic Acetohydrazone-Modified Phosphorus Dendrimers for Biomedical Applications: Gelation Studies and Nucleic Acid Loading.

In this work, we report the assemblage of hydrogels from phosphorus dendrimers in the presence of biocompatible additives and the study of their interactions with nucleic acids. As precursors for hydrogels, phosphorus dendrimers of generations 1⁻3 based on the cyclotriphosphazene core and bearing ammonium or pyridinium acetohydrazones (Girard reagents) on the periphery have been synthesized. The gelation was done by the incubation of dendrimer solutions in water or phosphate-buffered saline in the presence of biocompatible additives (glucose, glycine or polyethylene glycol) to form physical gels. Physical properties of gels have been shown to depend on the gelation conditions. Transmission electron microscopy revealed structural units and well-developed network structures of the hydrogels. The hydrogels were shown to bind nucleic acids efficiently. In summary, hydrogels of phosphorus dendrimers represent a useful tool for biomedical applications.

Key Aspects of Nucleic Acid Library Design for in Vitro Selection.

Nucleic acid aptamers capable of selectively recognizing their target molecules have nowadays been established as powerful and tunable tools for biospecific applications, be it therapeutics, drug delivery systems or biosensors. It is now generally acknowledged that in vitro selection enables one to generate aptamers to almost any target of interest. However, the success of selection and the affinity of the resulting aptamers depend to a large extent on the nature and design of an initial random nucleic acid library. In this review, we summarize and discuss the most important features of the design of nucleic acid libraries for in vitro selection such as the nature of the library (DNA, RNA or modified nucleotides), the length of a randomized region and the presence of fixed sequences. We also compare and contrast different randomization strategies and consider computer methods of library design and some other aspects.

Nuclease-resistant 63-bp trimeric siRNAs simultaneously silence three different genes in tumor cells.

We designed a multimeric nuclease-resistant 63-bp trimeric small-interfering RNA (tsiRNA) comprising in one duplex the sequence of siRNAs targeting mRNAs of MDR1, LMP2, and LMP7 genes. We show that such tsiRNA is able to suppress the expression of all the target genes independently and with high efficiency, acting via a Dicer-dependent mechanism. tsiRNA is diced into 42- and 21-bp duplexes inside the cell. tsiRNA-induced gene silencing is characterized by kinetics similar to that of canonical siRNA, while the silencing efficiency is significantly higher than that of canonical siRNA with the same sequence.

Structural rearrangements in mRNA upon its binding to human 80S ribosomes revealed by EPR spectroscopy.

The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5'- and 3'-terminal nucleotides and prone to form a stable homodimer (MR)2, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)2: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs.

Structural features of the interaction of the 3'-untranslated region of mRNA containing exosomal RNA-specific motifs with YB-1, a potential mediator of mRNA sorting.

We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3'-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA. With recombinant YB-1, it was found that SEPT14 RNA can bind up to 5 moles of protein per mole of RNA in a cooperative manner, which was shown to be mainly facilitated by the presence of the above motifs. RNA hairpins with motifs 1 and 2 competed with SEPT14 RNA for binding to the protein, whereas that with motif 3 was less competitive, in accordance with the affinity of YB-1 for these RNA hairpins. With YB-1-bound RNA, nucleotides protected from attack by hydroxyl radicals were revealed in all three motifs, although hairpins with motif 2 and especially with motif 1 contained many protected nucleotides outside the motifs, suggesting that the specific environments of these motifs contribute significantly to the YB-1 binding. An analysis of the environments of motifs 1-3 in the HEK293 cell mRNA 3' UTRs gained from RNA-seq data led us to conclude that the primary binding sites of YB-1 in the 3' UTRs are hairpins containing some part of the motif along with its specific surroundings; the consensus sequences of these hairpins were derived. Thus, our findings provide a new understanding of the structural basis of the interactions between YB-1 and mRNAs carrying the aforementioned motifs.

Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides.

In this review, we summarize the recent advances in the use of pyrene-modified oligonucleotides as a platform for functional nucleic acid-based constructs. Pyrene is of special interest for the development of nucleic acid-based tools due to its unique fluorescent properties (sensitivity of fluorescence to the microenvironment, ability to form excimers and exciplexes, long fluorescence lifetime, high quantum yield), ability to intercalate into the nucleic acid duplex, to act as a π-π-stacking (including anchoring) moiety, and others. These properties of pyrene have been used to construct novel sensitive fluorescent probes for the sequence-specific detection of nucleic acids and the discrimination of single nucleotide polymorphisms (SNPs), aptamer-based biosensors, agents for binding of double-stranded DNAs, and building blocks for supramolecular complexes. Special attention is paid to the influence of the design of pyrene-modified oligonucleotides on their properties, i.e., the structure-function relationships. The perspectives for the applications of pyrene-modified oligonucleotides in biomolecular studies, diagnostics, and nanotechnology are discussed.

Amphiphilic carbosilane dendrons as a novel synthetic platform toward micelle formation.

A novel family of amphiphilic ionic carbosilane dendrons containing fatty acids at the focal point were synthesized and characterized. They spontaneously self-assembled in aqueous solution into micelles both in the absence and presence of salt, as confirmed by surface tension, conductivity, and DLS measurements. Dendron based micelles have spherical shapes and increase in size on decreasing dendron generation. These dendritic micelles have been demonstrated to be able to form complexes with therapeutic macromolecules such as siRNA and show a high loading capacity for drugs such as procaine, suggesting their potential use as nanocarriers for therapeutics.